Bacterial secretion has long been recognized as an essential facet of microbial pathogenesis and human disease. One important but poorly understood system, which is ubiquitous among Gram-negative organisms, involves packaging cargo into small outer membrane derived vesicles (OMVs). Numerous virulence factors have been found to be transported in this way, and delivery by OMVs often results in increased potency. OMVs have also been implicated in the killing of host cells and competing bacteria, avoidance of and interference with the immune system, horizontal gene transfer mediating antibiotic resistance, biofilm formation, and trafficking small molecule communication signals. Remarkably, little is known about how these versatile structures are formed or how their cargo is selected and packaged. To address this, our team proposed the Bilayer-Couple Model where intercalation of self-produced small molecules into the outer membrane drives the induction of membrane curvature to initiate OMV formation. This is a biochemical/biophysical model that followed the discovery by our team and colleagues that the Pseudomonas Quinolone Signal (PQS) is packaged within and drives biogenesis of OMVs in Pseudomonas aeruginosa. In developing this model, we encountered a problem that is common in membrane biology: while all biological membranes contain asymmetric lipid distributions (leaflet vs. leaflet), it was impossible to generate a useful quantiy of in vitro liposomes matching these characteristics. Thus, weakly-relevant surrogates had to be used. Recently, our team has developed a novel approach for constructing synthetic asymmetric vesicles possessing a bilayer architecture that is more physiologically accurate than any other available system. Our approach utilizes microfluidic technology to build vesicles with controlled size, membrane asymmetry, uniformity, and luminal content. These vesicles are the ideal system to experimentally test the predictions of the Bilayer-Couple Model. To gain a greater physical insight in complement to experiments, we also propose to create the first-ever atomistic molecular dynamics and mesoscopic dissipative particle dynamics simulation of the bacterial outer membrane to discover the specific interactions between PQS and physiological-relevant asymmetric membranes. In particular, this model will help elucidate the detailed dynamics of PQS insertion into the outer membrane, its orientation PQS vs. surrounding lipids in the leaflet and whether its own physical properties direct its observed packaging into OMVs. Using leading edge experimental and computational tools, this proposal will address fundamental aspects of OMV formation, including (1) how PQS interacts with and alters the structure of the outer membrane, (2) whether these interactions are sufficient to initiate OMV formation, and (3) whether PQS itself may contribute to its accumulation in OMVs as cargo. The fundamental mechanistic foundations established through this study will have implications in many aspects of health research, potentially enabling applied topics such as vaccine development and drug delivery, for which OMVs are rapidly becoming exciting candidates.